GSM6318439: Flybrain_rehydrated_2; Drosophila melanogaster; RNA-Seq
For each sample, 24 central brains from mixed sexes (1:1) were individually dissected in ice-cold DPBS (Gibco, 14190–086) and immediately transferred into 1 mL toxin-supplemented Schneider's medium (tSM: Gibco, 21720–001 + 50 mM d(-)-2- amino-5-phosphonovaleric acid, 20 mM 6,7-dinitroquinoxaline-2,3-dione and 0.1 mM tetrodotoxin) on ice. Brains were washed once with 1 mL tSM and incubated in tSM containing 1.11 mg/mL papain (Sigma, P4762) and 1.11 mg/mL collagenase I (Sigma, C2674). Brains were washed once more with tSM and subsequently triturated with flame-rounded 200 mL pipette tips. Dissociated brains were resuspended into 1 mL PBS + 0.01% BSA and filtered through a 10 mm CellTrix strainer (Sysmex, 04-0042-2314). Cell concentration was measured using a disposable Fuchs-Rosenthal haemocytometer (VWR, 631–1096) under a Leica DMIL LED Fluo microscope. A typical preparation from 24 brains yielded ~600,000 cells. mRNA barcoding and library preparation were performed using the Chromium Single Cell 3′ Reagent Kit v3 (10x Genomics), following manufacturer's instructions. For each sample, we targeted an 8,000 cell recovery. Libraries were sequenced with NovaSeq 6000 (Illumina)